U.S. patent number 5,218,079 [Application Number 07/603,111] was granted by the patent office on 1993-06-08 for soluble melanin.
This patent grant is currently assigned to Yale University. Invention is credited to Seth J. Orlow, John M. Pawelek.
United States Patent |
5,218,079 |
Pawelek , et al. |
June 8, 1993 |
Soluble melanin
Abstract
A melanin that is soluble in an aqueous solution at a pH of at
least 5 to 9 at a temperature of 0.degree. to 100.degree. C. The
melanin is further characterized by being capable of being filtered
through at least a 0.45 micron size filter. Still further, the
melanin is characterized by having a molecular weight of greater
than 10,000 kilodaltons. The melanin is useful for providing a
naturally-appearing tan to mammalian skin and hair. Such melanin
can be produced by combining dopachrome and 5,6-dihydroxyindole (or
allowing dopachrome to spontaneously form 5,6-dihydroxyindole) and
an appropriate enzyme or by combining 5,6-dihydroxyindole and
5,6-dihydroxyindole-2-carboxylic acid or by incubating
5,6-dihydroxyindole-2-carboxylic acid alone. The melanin is also
useful for providing a sun-screen to mammalian skin and hair.
Inventors: |
Pawelek; John M. (Hamden,
CT), Orlow; Seth J. (Long Island City, NY) |
Assignee: |
Yale University (New Haven,
CT)
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Family
ID: |
27061961 |
Appl.
No.: |
07/603,111 |
Filed: |
October 25, 1990 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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525944 |
May 18, 1990 |
|
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Current U.S.
Class: |
528/206; 424/59;
424/63; 435/41 |
Current CPC
Class: |
A61K
8/72 (20130101); A61K 31/785 (20130101); A61Q
17/04 (20130101); C09B 69/104 (20130101); C12P
17/00 (20130101); C12P 17/10 (20130101); A23L
5/43 (20160801); A23L 5/47 (20160801); Y10S
514/937 (20130101); Y10S 514/938 (20130101); Y10S
514/939 (20130101); Y10S 514/969 (20130101) |
Current International
Class: |
A23L
1/27 (20060101); A23L 1/275 (20060101); A61K
31/74 (20060101); A61K 31/785 (20060101); A61K
8/72 (20060101); A61Q 17/04 (20060101); C09B
69/00 (20060101); C12P 17/10 (20060101); C12P
17/00 (20060101); C09B 69/10 (20060101); C08G
063/06 () |
Field of
Search: |
;424/59,63 ;528/206
;435/41 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Korner & Gettins BA 81(1):5362 (1985). .
Korner & Pawelek BA 71(1):3755 (1981). .
Orlow et al., Pigment Cell Research 5:113-121 (1992). .
American Scientist, Mar.-Apr. 1982, vol. 70, No. 2. .
Chem. J. (1974) 143, 207-217, "Isolation of Oligomers of
5,6-Dihydroxyindole-2-Carboxylic Acid From the Eye of the Catfish",
pp. 207-217. .
Biochemistry 1988, 27, 6156-6159, "Function of Dopachrome
Oxidoreductase and Metal Ions in Dopachrome Conversion in the
Eumelanin Pathway". .
Biochemical et Biophysica Acta 925 (1987) 203-209, "Effect of Metal
Ions on the Rearrangement of Dopachrome"..
|
Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Witz; Jean C.
Attorney, Agent or Firm: Sprung Horn Kramer & Woods
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part application of
application Ser. No. 07/525,944, filed May 18, 1990, now pending.
Claims
What is claimed is:
1. A method of producing soluble melanin comprising in an aqueous
reaction medium polymerizing [5,6-dihydroxyindole-2-carboxylic
acid] a dihydroxyindole-carboxylic acid in the presence of
oxygen.
2. A method of producing a soluble melanin according to claim 1,
wherein the reaction mixture further contains
5,6-dihydroxyindole.
3. A method according to claim 1, wherein the reaction mixture
further comprises indole-5,6-quinone.
4. A method according to claim 1, wherein the reaction mixture
further comprises melanochrome.
5. A method according to claim 1, wherein the reaction mixture
further comprises indole-5,6-quinone and melanochrome.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns the synthesis of soluble forms of
melanin and their composition, and methods of using such
compositions to provide a naturally-appearing tan to mammalian skin
and hair and to provide a sun-screen.
2. Background Information
In biology, melanins are heteropolymers consisting of L-dopa and
its enzymatic derivatives. They are ubiquitous in living organisms
and are produced throughout the zoological and botanical phyla. In
mammalian skin, melanins are produced through enzymatic processes
in specialized cells known as "melanocytes". Melanins are the
pigments of mammalian skin and hair.
Mammalian melanins are highly insoluble and can be dissolved
(solubilized) only through non-physiological treatments such as
boiling in strong alkali, or through the use of strong oxidants
such as hydrogen peroxide. Tyrosinase, a key enzyme in the melanin
biosynthetic pathway, can catalyze the formation of melanin in a
test tube using L-tyrosine, L-dopa or 5',6'-dihydroxyindole as
substrates, however, the product is an insoluble precipitate as
described above.
Ito, "Reexamination of the Structure of Eumelanin", Biochimica et
Biophysica Acta, 883, 155-161, 1986, mentions natural melanin may
be a polymer of 5,6-dihydroxyindole and
5,6-dihydroxyindole-2-carboxylic acid. Ito, however, does not teach
or suggest combining these chemicals to form melanin.
Ito and Nicol, "Isolation of Oligimers of
5,6-Dihydroxyindole-2-carboxylic Acid from the Eye of the Catfish",
Biochemical Journal, 143, 207-217, 1974, mention that oligimers of
5,6-dihydroxyindole-2-carboxylic acid exist in nature, for example
in the tapetum lucidum of the sea catfish (Arius felis). Ito and
Nicol, however do not teach or suggest that these structures could
be used as a form of soluble melanin.
Palumbo, d'Ischia, Misuraca, and Prota, "Effect of metal ions on
the rearrangement of dopachrome", Biochimica et Biophysica Acta.
925, 203-209, 1987, mention that the metal ions CU.sup.2+,
Ni.sup.2+, and CO.sup.2+ are effective in inducing the
non-decarboxylative rearrangement of dopachrome at physiological pH
values, leading mainly to the formation of
5,6-dihydroxyindole-2-carboxylic acid. They suggest that when
considered in the light of the known metal accumulation in
pigmented tissues, their results provide a new entry into the
regulatory mechanisms involved in the biosynthesis of melanins.
Palumbo et al, however, do not teach or suggest that such metal
ions could be used to affect the color or formation of soluble
melanin. Likewise, Leonard, Townsend, and King, "Function of
Dopachrome Oxidoreductase and Metal Ions in Dopachrome Conversion
in the Eumelanin Pathway",. Biochemistry, 27, 6156-6159, 1988,
present similar results to those of Palumbo et al regarding metal
ions and the formation of 5,6-dihydroxyindole-2-carboxylic acid
from dopachrome. Like Palumbo et al, Leonard et al also do not
teach or suggest that such metal ions could be used to affect the
color for formation of soluble melanin.
Many reports exist exploring the role of sulfhydryl compounds such
as cysteine or glutathione in determining the red or yellow colors
in melanins (see review by Pawelek and Korner, "The Biosynthesis of
Mammalian Melanin", American Scientist, 70, 136-145, 1982). However
these reports do not teach or suggest that said sulfhydryl
compounds could be used to influence the colors of soluble
melanin.
It would be of commercial value to have forms of melanin which are
soluble at physiological pH and temperature. Such melanins could be
applied evenly to mammalian skin and hair in appropriate vehicles
without any of the caustic side-effects arising from the harsh
reagents needed to solubilize precipitated melanins.
Such solubilized melanins could impart a naturally-appearing tan to
mammalian skin and hair. Solubilized melanins would also be
effective as sun-screens, since melanins are the chemicals in the
skin which absorb ultraviolet radiation and thus provide protection
from its harmful effects, such as premature skin aging and the
occurrence of skin cancers.
SUMMARY OF THE INVENTION
It is the object of the present invention to provide solubilized
forms of melanin at physiological pH and temperatures.
It is another object of the present invention to provide
compositions and methods for applying such melanins to mammalian
skin and hair to provide a naturally-appearing tan.
It is another object of the present invention to provide
compositions and methods for applying such melanins to mammalian
skin and hair to provide a sun-screen.
The above objects and other objects, aims, and advantages are
satisfied by the present invention. The present invention relates
to a melanin that is soluble in aqueous solution, e.g., water or an
aqueous buffered solution, at a pH of at least 5 to 9, preferably
6.5 to 7.5, at a temperature of 0.degree. to 100.degree. C. The
soluble melanin is further characterized by being capable of being
filtered through at least a 0.45 micron size filter. The solubility
of the melanin is in large part due to the abundance of
carboxyl-groups.
The present invention also concerns a method of producing
solubilized melanin comprising combining in a reaction mixture
dopachrome and one or more enzymes derived from biological cells or
tissues which have a pigmentary system. 5,6-dihydroxyindole may be
added to the dopachrome and enzyme(s) or dopachrome may be allowed
to spontaneously form 5,6-dihydroxyindole before adding the
enzyme(s). Alternatively, the reaction mixture may comprise
5,6-dihydroxyindole-2-carboxylic acid alone or in a mixture with
5,6-hydroxyindole, in which case enzymes are not necessary and the
reaction occurs in the presence of oxygen.
The color of the soluble melanin can be varied between black,
brown, red and yellow by altering the contents of the reaction
mixtures, for example by adding sulfhydryl containing compounds, or
various metals such as, but not limited to, CU.sup.2+, Ni.sup.2+,
and CO.sup.2+ or by altering the pH of the reaction mixtures.
The basis for the solubility of the melanin is in a large part due
to the high degree of carboxyl groups present in the molecule, said
carboxyl groups being incorporated a part of the
5,6-dihydroxyindole-2-carboxylic acid precursor. Compounds similar
to 5,6-dihydroxyindole-2-carboxylic acid could substitute in
providing said carboxyl groups and could therefore also act as
precursors to soluble melanin.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a photograph showing the enzymatic formation of
soluble melanin according to the invention. Non-enzymatically
formed soluble melanin is similar in composition to that seen in
the tubes labelled "DI Complex".
FIG. 2 depicts a graph showing the optical density (O.D.) of
soluble melanin according to the invention at wavelengths greater
than 300 nm.
The soluble melanin was synthesized non-enzymatically by mixing
5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid
(DHICA). The closed diamonds represent a fresh, non-incubated
mixture of DHI and DHICA where no soluble melanin was present. The
open squares represent the same mixture incubated 18 hours at room
temperature in the presence of oxygen during which time soluble
melanin was synthesized.
The high absorbance peaking between 310-320nm is characteristic of
the presence of DHICA in the melanin. The broad absorbance over the
range from 400-600nm is due in the presence of visible color,
characteristic of soluble melanins. Shown here are absorbance
spectra in the "ultraviolet A" range and higher. Not shown are the
strong absorbance spectra of the soluble melanin in the ultraviolet
B and C ranges.
FIG. 3 depicts a graph showing the optical density (O.D.) of
soluble melanin according to the invention at wavelengths greater
than 300 nm. The soluble melanin was synthesized non-enzymatically
as in FIG. 2 under either aerobic (closed diamonds) or anaerobic
conditions (open square). It can be seen that oxygen increases the
amount of absorbance in the 400-600nm range, i.e., the amount of
visible color.
FIG. 4 depicts a graph showing the optical density (O.D.) of
soluble melanin according to the invention at wavelengths greater
than 300 nm. The melanin was synthesized either non-enzymatically
by mixing DHI and DHICA (closed diamonds) as described in FIGS. 2
and 3; or enzymatically by mixing dopachrome isomerase enzyme
complex (DI) with dopachrome and DHI (open diamonds), or by mixing
DI with only DHI (open squares), or by mixing DI with DHI plus
DHICA (closed squares). The results demonstrate that enzymatic and
non-enzymatic methods for synthesizing soluble melanin yield
comparable products. They also demonstrate the necessity of DHICA
or a compound similar to DHICA in the reaction mixtures.
FIG. 5 depicts a graph showing the optical density of soluble
melanin synthesized non-enzymatically at pH 7. The reaction
mixtures were comprised of a mixture of DHICA and DHI (closed
diamonds) or DHICA alone (open squares). The results demonstrate
that at ph 7 more visible melanin (400-600 nm) is synthesized with
a mixture of DHI and DHICA than with DHICA alone.
FIG. 6 depicts a graph showing the optical density of soluble
melanin synthesized non-enzymatically at pH 8. The reaction
mixtures are otherwise the same as those in FIG. 5. The results
demonstrate that at pH 8, DHICA alone can serve as an efficient
precursor to the formation of soluble melanin and in fact is
somewhat superior to a mixture of DHICA and DHI.
FIG. 7 depicts a graph showing a pH titration curve as increasing
amounts of acetic acid are added to a solution of 2 mM soluble
melanin.
FIG. 8 depicts a graph showing the precipitation of soluble melanin
during the pH titration shown in FIG. 7. For each point, acetic
acid was added and the solution was allowed to sit at room
temperature before being filtered through a 0.45 micron filter. The
optical density of the filtrate was then determined at 500 nm. The
soluble melanin begins to precipitate below pH4, i.e. as the pK of
the carboxyl groups is reached. The results are consistent with the
solubility of the melanin being determined by the number of
non-protonated carboxyl groups present in the molecule.
DETAILED DESCRIPTION OF THE INVENTION
The soluble melanin of the invention remains in aqueous solution,
at neutral pH (e.g., pH of 5 to 9, preferably 6.5 to 7.5), for long
periods of time, e.g., indefinitely, at temperatures of 0.degree.
C. to 100.degree. C., e.g., room temperature. The soluble melanin
according to the invention is further characterized by remaining
soluble upon freezing/thawing. The inventive soluble melanin is
also characterized by being capable of being filtered through at
least a 0.45 micron size filter. The soluble melanin according to
the invention can be precipitated below pH4.
Following synthesis, the soluble melanin cannot be dialyzed through
a semi-permeable membrane which allows the passage of molecules
less than a molecular weight of approximately 10,000 daltons.
Therefore the soluble melanin according to the invention is of a
molecular weight greater than 10,000 daltons, however, this is not
an essential characteristic for its usefulness. The soluble melanin
can be lyophilized to a dry powder form and then reconstituted to
its soluble form with distilled water or suitable aqueous solvents,
e.g., sodium phosphate 0.1M or sodium chloride 0.1M.
The soluble melanin according to the invention can be prepared
non-enzymatically (synthetically) or enzymatically.
The enzymatic preparation according to the invention comprises
combining in a reaction mixture a substrate, i.e., dopachrome, and
one or more enzymes derived from biological cells or tissues which
contain a pigmentary system and more particularly have the ability
to produce melanin.
In the non-enzymatic preparation according to the invention, the
reaction mixture comprises as a substrate
5,6-dihydroxyindole-2-carboxylic acid (DHICA) alone or a mixture of
DHICA and 5,6-dihydroxyindole. Suitable analogs of DHICA, i.e.
similar structures containing carboxyl groups, maybe substituted in
the reaction. The enzymatic or nonenzymatic reaction mixtures may
still further comprise as a substrate indole-5,6-quinone and/or
melanochrome. Metal ions and sulfhydryl-containing compounds may be
included.
The individual components of the substrate, be it one component,
i.e., dopachrome, as in the enzymatic preparation, or more than one
component as in the nonenzymatic preparation, preferably will be in
an amount of 0.01 to 5.0 millimolar. Stated otherwise, when more
than one component is used, the components, i.e., a mixture of
5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid, will
preferably be in equal proportions or near to equal
proportions.
The combining of substrate and enzymes or substrates in the
reaction mixture is preferably conducted at a temperature of
15.degree. C. to 37.degree. C.
It is preferred in both the enzymatic and nonenzymatic preparation
that oxygen, e.g., air or pure oxygen, be present. This is
especially true for the nonenzymatic preparations.
Structural formulas and the relationship among some of the above
described compounds are depicted as follows: ##STR1##
From the above it is seen that dopa quinone, leuco dopachrome,
dopachrome, DHICA, 5,6-dihydroxyindhole, indole-5,6-quinone, and
melanochrome are all derivatives of dopa. Dopa itself is a
derivative of tyrosine, so the above compounds are also derivatives
of tyrosine (see Scheme II). Scheme I shows that dopachrome can
give rise to 5,6-dihydroxyindole in a spontaneous non-enzymatic
reaction, or it can give rise to DHICA in an enzymatically
catalyzed reaction. The enzyme which catalyzes dopachrome to DHICA
is named dopachrome isomerase and may indeed be the enzyme
responsible for soluble melanin formation. The enzyme may also be a
part of a complex comprising tyrosinase, dopachrome isomerase,
glycoprotein 75, MSH receptor and other unknown proteins.
The above described enzymes are described in the following
papers:
tyrosinase: Ann Korner and John Pawelek, "Mammalian Tyrosinase
Catalyzes Three Reactions in the Biosynthesis of Melanin", Science,
217:1163-1165, 1982;
dopachrome isomerase: John Pawelek, "Dopachrome Conversion Factor
Functions as an Isomerase", Biochemical and Biophysical Research
Communications, 166:1328-1333, 1990;
glycoprotein 75: Timothy M. Thomson, M. Jules Mattes, Linda Roux,
Lloyd Old and Kenneth O. Lloyd, "Pigmentation-associated
Glycoprotein of Human Melanomas and Melanocytes: Definition with a
Mouse Monoclonal Antibody", J. Invest. Derm., 85:169-174, 1985:
MSH receptor: Seth J. Orlow, Sara Hotchkiss, and John M. Pawelek,
"Internal Binding Sites for MSH: Analyses in Wild-Type and Variant
Cloudman Melanoma Cells", J. Cellular Physiology, 142:129-136,
1990.
The enzyme may also include dopachrome isomerase and one or more of
glycoprotein 75, MSH receptor and tyrosinase.
The enzyme may take the form of one or more individual enzymes or a
complex of enzymes including tyrosinase, dopachrome isomerase,
glycoprotein 75, MSH receptor and one or more additional enzymes
which are distinct from the aforesaid four described enzymes, but
which are capable of catalyzing the synthesis of soluble
melanin.
The soluble melanin according to the present invention can be
admixed with a physiologically acceptable carrier to form a
composition.
Physiologically acceptable carriers useful in the practice of the
invention are known in the art and non-limiting examples of such
carriers include, for controlled release--microcapsules comprising
carboxymethylene copolymers; for transdermal release--acrylamides
and for topical application--cosmetic bases.
In addition, if desired, the composition according to this
embodiment comprises at least one additive selected from the group
consisting of solvents, fragrances, sunscreening agents,
preservatives and chelating agents.
Cosmetic bases useful in the practice of the invention are well
known and include lotions, creams, ointments and dusting powders.
Examples thereof may be found in, e.g., U.S. Pat. Nos. 4,228,151;
4,282,206 and 2,949,403.
Solvents for use in accordance with the invention include, for
example, ethanol, isopropyl alcohol, benzyl alcohol, oils, for
example, ground nut oil, distilled and/or deionized water,
physiological saline solution and the like. The specific solvent
chosen will depend on the method of application.
It may also be desirable to add a preservative to the inventive
compositions if they are to be used for topical applications. The
preferred mode of administration of the inventive compositions is
topical administration. Still further, the soluble melanin of the
present invention may be combined with substances that stimulate
the pigmentary system under conditions of low levels of UV
light.
Preservatives are well known and may be exemplified by
methylparaben, "DOWACIL 2000" and propylparaben.
If desired, in order to reduce the acidity or basicity of the
inventive compositions, bases, acids or buffers may be added
thereto in accordance with the knowledge of the art.
The concentration of soluble melanin in an aerosol, cream, lotion
or other composition is preferably 0.01 mg/ml to 1.0 mg/ml.
Solutions have different colors depending on the concentration of
"chromophore" dissolved in them. For example, a deep red solution
will appear orange or pink when diluted with more solvent, but no
additional chromophore. In the case of the soluble melanin, when it
is dissolved at a fairly high concentration in water, e.g., 0.5
mg/ml, it appears brown-black in color. When more water is added so
that the concentration of the soluble melanin is reduced to, e.g.,
0.1 mg/ml, the solution appears golden in color. It is not believed
that diluting the material changes any shift in the absorbance
spectrum, rather it is believed to be a visual perception.
For the nonenzymatic preparation, the
5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihdyroxyindole may
be maintained separately, for example, in microspheres, or in
separate tubes or containers, until being mixed together on the
skin of a mammal, e.g., human.
The invention will now be described with reference to the following
non-limiting examples.
EXAMPLE 1
Source of Enzymes
The enzyme preparation for the synthesis of soluble melanin can be
obtained from any biological cells or tissues which have the
ability to produce melanin, for example:
(1) extracts of vertebrate skins from horses, cattle, sheep, pigs,
or any other such mammalian source; extracts of skins of fish,
amphibia, reptiles, birds, or any other such vertebrate source;
(2) extracts of any botanical source which has the ability to
produce melanin, such as mushrooms, potatoes, bananas, or any other
such botanical source;
(3) extracts of invertebrate organisms such as worms, arthropods,
or any such invertebrate source which has the ability to produce
melanin;
(4) extracts of any single-cell organisms which have the ability to
produce melanin, but which do not necessarily fall into a category
of zoological or botanical, such as bacteria and protozoans;
(5) extracts of any organisms in which genes for enzymes producing
soluble melanin have been genetically cloned; and
(6) culture media of any organisms or cells which secrete enzymes
for producing soluble melanin--such organisms or cells may or may
not express cloned genes for the enzymes.
Furthermore, the source of biological material for the isolation of
enzymes to produce soluble melanin does not necessarily have to be
actively synthesizing melanin in vivo. A pigmentary system is
defined as all or part of a group of enzymes which can recognize as
their substrates precursors and/or intermediates in the melanin
biosynthetic pathway. Some biological sources contain a pigmentary
system, but do not synthesize melanin, however, extracts derived
from them can produce soluble melanin. These biological systems, in
their living state, are referred to as "amelanotic", or
"non-pigmented". Many albino organisms fall into this category,
i.e., they possess an incomplete or inhibited pigmentary system and
do not make melanin in vivo, however, extracts from some albino
organisms contain enzymes that can produce soluble melanin. Such
organisms are also potential sources of enzymes for the production
of soluble melanins.
EXAMPLE 2
Preparation of Enzymes
The enzymes for production of soluble melanin can be isolated from
an extract of an appropriate biological source, or, from the
culture media should the enzymes be secreted into the media by the
source (see Example 1). Extracts are prepared by lysing the cells
of the biological source through procedures such as homogenization
(e.g., in a common kitchen "blender", in appropriate glass, metal,
or plastic tissue homogenizers); such as freeze-thawing in a
hypotonic solution such as water; or by any means which disrupt the
cellular wall or plasma membrane of the biological source. In cases
where the enzymes for producing melanin are themselves insoluble
(e.g., in a particulate form within the cells), it may be necessary
to lyse the cells in the presence of a non-ionic detergent such as
"TRITON X-100", "CHAPS", or "TWEEN 80", or an organic solvent such
as acetone or ethanol, or any other solvents which solubilize the
enzymes, without destroying their ability to produce soluble
melanin.
The following procedures may be useful, but are not mandatory for
the preparation:
Extracts containing the enzymes in a soluble form can be clarified
by filtration through such material as gauze or filter paper; or by
centrifugation; or by "settling" through the use of natural
gravitational force.
Extracts can be further clarified by mixing them with calcium
phosphate (also known as "hydroxylapatite") which is itself
insoluble, but which attaches to many molecular structures in
cells, but does not attach to the enzymes for preparation of
soluble melanins. The calcium phosphate and its attached molecules
can be removed from the extract by filtration, centrifugation, or
gravitational settling as described above. Although calcium
phosphate is useful in this regard, the procedure is not restricted
to the use of this agent only. Any insoluble compound which
attaches to molecular structures other than the enzymes in question
and does not destroy the activity of the enzymes can be
employed.
Extracts can be further clarified by mixing them with an anion
exchange agent such as diethylaminoethyl cellulose. At conditions
around neutral pH (e.g., pH 6.5-7.5) and low buffer concentration
(e.g., 5-10 millimolar sodium phosphate), the enzymes for
production of soluble melanin will attach to such an anion exchange
agent, while many other molecular structures will not. The enzymes
in question can then be eluted from the anion exchange agent by
increasing the salt concentration (e.g., by adding 0.4 molar sodium
chloride, or by using a gradient of sodium chloride from 0 molar to
0.4 molar). Although diethylaminoethyl cellulose, sodium phosphate,
and sodium chloride are useful in this regard, the procedure is not
restricted to the use of these agents only. Any anion exchange
resin, buffer, or salt which does not destroy the activity of the
enzymes in question can be employed. The principle is the same.
Extracts can be further clarified by mixing them with an agent
which attaches to glycoproteins, such as wheat germ
lectin-sepharose. In mammalian cells, the enzymes in question are
glycoproteins and therefore attach to such lectins, while many
other molecular structures do not. The enzymes in question can then
be eluted from the lectin by mixing with an appropriate sugar such
a N-acetylglucoseamine, which causes a displacement of the enzymes
from the lectin. Although wheat germ lectin-sepharose, and
N-acetylglucoseamine are useful in this regard, the procedure is
not restricted to the use of these agents only. Any appropriate
lectin or sugar which does not destroy the activity of the enzymes
in question can be employed. The principle is the same.
Extracts can be further clarified by applying them to columns
containing molecular sieves such as Sephadex columns, or high
pressure liquid chromatography columns containing molecular sieves.
The enzymes in question can be eluted from such columns with
non-destroying buffers according to their molecular weights and can
be thereby separated from other molecular structures with differing
molecular weights. Although Sephadex columns and various HPLC
resins are useful molecular sieves, the procedure is not restricted
to the use of these agents only. Any appropriate method for
separation of molecules according to their molecular weights which
does not destroy the activity of the enzymes in question can be
employed. When each of the above procedures is carried out in the
sequence listed, a preparation is obtained which contains the
following: tyrosinase, dopachrome isomerase, a protein designated
"glycoprotein 75" or "gp75", which exhibits catalase activity, and
a protein designated "MSH receptor". That is, these four known
proteins co-purify through the above procedures. Analyses by
polyacrylamide gel electrophoresis indicate that there may be ten
or more proteins in total. It is not presently known which protein
or combination thereof catalyzes the synthesis of soluble melanin,
although the synthesis can occur in the presence of phenylthiourea,
a potent inhibitor of tyrosinase, suggesting that tyrosinase may
not be necessary.
EXAMPLE 3
Enzymatic Synthesis of Soluble Melanin
Soluble melanin is prepared enzymatically by mixing the enzymes,
isolated and purified as described above, with a solution
containing dopachrome and 5,6-dihydroxyindole. The enzymes and the
substrates are allowed to incubate in a non-destroying buffer
(e.g., 0.1 molar sodium phosphate, pH 6.5-7.5) at ambient
temperature or any suitable non-destroying temperature which allows
for the reaction to occur (e.g., 15.degree.-37.degree. C.), until
soluble melanins begin to appear (e.g., 3-6 hours). The reaction
can be monitored visually or with the use of a spectrophotometer
set in the visual spectrum (e.g., 400 millimicrons). When the
reaction has reached completion, the salts from the buffer can be
removed by dialysis and the soluble melanin can be stored at room
temperature, frozen, or as a crystal or powder obtained through
such procedures as natural evaporation or lyophilization. It is
useful, but not mandatory, to enzymatically synthesize soluble
melanin in the presence of a tyrosinase inhibitor such as
phenylthiourea, because tyrosinase, which is occasionally present
in the enzyme preparation, can cause the formation of insoluble
melanin. Although phenylthiourea is a useful tyrosinase inhibitor
because it does not inhibit or destroy the enzymes in question, the
procedure is not restricted to phenylthiourea and any such
tyrosinase inhibitor can be employed.
Soluble melanin prepared by the above procedure is golden-brown in
color and absorbs widely throughout the ultraviolet and visible
spectra (e.g., 220 to 700 millimicrons). The color can vary from
brown-black in very concentrated solutions, to golden in more
dilute solutions. The addition of sulfhydryl-containing compounds
such as cysteine or glutathione can impart a reddish color to the
soluble melanins.
FIG. 1 depicts the enzymatic formation of soluble melanin. The
contents of the three tubes on the right were filtered through a
0.45 micron filter, otherwise they were identical in composition to
the contents of the three tubes on the left. Considering the three
tubes on the left:
1) The far left-hand tube contained a mixture of dopachrome, dopa
and 5,6-dihydroxyindole. It also contained the buffer used to
dissolve the enzymes (sodium phosphate 5 mM, pH 6.8, containing 20%
glycerol vol/vol), but the enzymes themselves were not added.
2) The second tube from the left contained the same mixture as in
the left-hand tube, but in addition it contained the enzymes
dissolved in their buffer. The enzymes are referred to as "DI
Complex".
3) The third tube from the left was identical to the middle tube,
but also contained trypsin, a potent proteolytic enzyme, at a
concentration of 0.5 mg/ml.
The three tubes on the left were incubated at room temperature for
6 hours before half their contents were removed and filtered as
described above into the three right-hand tubes.
It can be seen that with buffer only, all the black melanin which
formed was trapped on the filter, i.e., insoluble. When the DI
Complex was added, the golden-brown melanin was completely
filterable, i.e., soluble. When trypsin was present, no soluble
melanin was formed, indicating that the soluble melanin production
was catalyzed by a protein (enzyme). Melanins prepared
non-enzymatically are similar in appearance to those seen in the
tubes labelled "DI Complex".
The soluble melanins provided in Example 3 have the following
characteristics:
(1) are greater than molecular weight 10,000,
(2) are stable to boiling,
(3) are stable to freezing,
(4) can be filtered through a filter at least as small as 0.45
microns,
(5) are soluble in water at a pH range of at least 6.5 to 7.5 at
temperatures from 0.degree. to 100.degree. C.,
(6) can be precipitated below pH 4,
(7) can be lyophilized to a crystal/powder form which can be
redissolved in water,
(8) vary from brown-black to golden in color depending on
concentration,
(9) absorb throughout the ultraviolet and visible spectra and
(10) can be prepared in red and yellow forms with the addition of
sulfhydryl-containing compounds and various metal ions.
EXAMPLE 4
Non-Enzymatic Synthesis of Soluble Melanin
Soluble melanin can be prepared in a nonenzymatic reaction by
mixing 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and
5,6-hydroxyindole (DHI) in the presence of oxygen in a
non-destroying buffer (e.g., 0.1 molar sodium phosphate, pH 6.5 to
7.5) or by incubating DHICA alone at ambient temperature of any
suitable non-destroying temperature which allows the reaction to
occur (e.g., 15.degree. to 37.degree. C.), until soluble melanin
begins to appear (e.g., 3 to 6 hours). The soluble melanins thus
formed is indistinguishable from that which is formed enzymatically
as described in Example 3.
The difference between enzymatic and non-enzymatic synthesis of
soluble melanin is that in the enzymatic synthesis,
5,6-dihydroxyindole-2-carboxyl acid is produced from dopachrome by
the enzyme dopachrome isomerase, and 5,6-dihydroxyindole is
produced spontaneously from dopachrome, while in the non-enzymatic
synthesis, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and
5,6-dihydroxyindole are mixed directly to form soluble melanin or
DHICA is incubated alone. In both the enzymatic and non-enzymatic
procedures, the reaction is greatly enhanced by the presence of
oxygen.
EXAMPLE 5
Spectrophotometric Quantitation of Soluble Melanin
Melanin was synthesized using a mixture of L-dopa, dopachrome,
dihydroxyindole, and dihydroxyindole-2-carboxylic acid at
concentrations of approximately 0.4 mg/ml dissolved in sodium
phosphate, 0.1M, pH 6.8. The "DI Complex" enzymes purified
approximately 2,000 fold from 0.5 grams mouse melanoma tissue (see
Example 3) were dissolved in buffer (sodium phosphate, 5 mM, pH
6.8) and incubated in a 4 ml reaction mix with the above substrates
at room temperature for 5 hours. The control reaction had buffer
only, with no enzymes added. In both reactions, melanin formation
occurred, but when the enzymes were present, the melanin could be
filtered through a 0.45 micron filter, whereas in the presence of
buffer only, the melanin was insoluble and could not be filtered.
Solutions were diluted 50 fold before measuring the optical
density. A photographic representation of this experiment is seen
in FIG. 1.
FIGS. 2 to 4 show the optical spectrum of soluble melanin according
to the present invention. In FIGS. 2 to 4, the melanin exhibited a
peak optical density (O.D.) at a wavelength of 310-320 nm. The
spectra and O.D. were the same after the melanin had incubated
under sterile conditions for 2 months at room temperature
(20.degree. C.), and whether or not the melanin was dialyzed or
filtered through a 0.45 micron filter.
Note in FIG. 4 that relatively little soluble melanin is made when
DHI plus DI are mixed together in the absence of any other added
substrates. Note also, when DHI plus DHICA are mixed together they
make as much soluble melanin as when they are mixed additionally
with DI, i.e., DI is not necessary in this case. Finally, note that
a mixture of dopachrome and DHI in the absence of DI results in an
insoluble precipitate (not shown here), but in the presence of DI
results in the synthesis of soluble melanin. This is because DI
converts dopachrome to DHICA which then in turn combines with DHI
in the presence of oxygen to form soluble melanin. In the absence
of DI, dopachrome spontaneously converts to DHI and a precipitate
(insoluble melanin) forms (see Scheme I hereinabove).
FIGS. 5 through 8 are described hereinabove.
TABLE 1 ______________________________________ Spectrophotometric
Quanititation of Soluble Optical Density at Wavelength 320 nm
Synthetic Route Before Filtration After Filtration
______________________________________ Buffer only 1.22 .024 (no
enzymes added) Enzymes added 0.982 0.966
______________________________________
EXAMPLE 6
Transdermal Release Composition
An admixture is prepared comprising the following:
______________________________________ Parts by weight
______________________________________ Acrylamide copolymer 20
(e.g., "polytrap FLME 203") soluble melanin, as prepared 5
according to Examples 3 or 4 Alcohol 74.9 and a fragrance 0.1
______________________________________
The above mixture is applied to the skin, once a day, preferably in
the morning, for two to four weeks.
EXAMPLE 7
Tanning Oil
A. An admixture is prepared by adding in the order indicated:
______________________________________ Parts by weight
______________________________________ decylolcate 25.0 isopropyl
myristate 15.0 and propylene glycol dicaprylate/ 5.0 dicaprate
mineral oil 54.85 ______________________________________
B. An admixture is prepared by adding 0.01 pbw soluble melanin,
e.g., as prepared as described in Example 3 or 4, to 0.01 pbw of
"SOLERTAN PB-10" (a poly(propylene glycol) lanolin ether).
C. The admixture of part B is added to the admixture of part A and
the resultant admixture is mixed until homogeneous.
D. The composition of part C is applied to the skin once or twice
daily for two to four weeks.
EXAMPLE 8
Suntanning Lotion
An admixture was prepared containing the following:
______________________________________ Parts by weight
______________________________________ ICI G-1800 5.0 (e.g.,
poly[oxyethylene]21 stearyl ether) isopropyl myristate 10.0
preservative 0.1 stearyl alcohol 2.0 2-hydroxy-3,3,5- 8.0
trimethylhexyl ester of benzoic acid butylated hydroxyanisole 0.05
______________________________________
The above mixture is heated to 70.degree. C. and 60 parts by weight
of water, preheated to 70.degree. C. is added thereto. the
resultant mixture is stirred and allowed to cool to room
temperature.
To the above mixture is then added a 1% citric acid solution, QS,
to achieve a pH of 5.0 after which 0.01 parts by weight of a
soluble melanin, for example, as prepared according to Example 3 or
Example 4, is added, as well as sufficient deionized water to yield
100 parts by weight of lotion.
The above lotion is applied to the skin one-half (1/2) hour prior
to exposure to the sun. After swimming, sweating or toweling, as
well as after each hour of exposure, the lotion is reapplied.
It will be appreciated that the instant specification is set forth
by way of illustration and not limitation, and that various
modification and changes may be made without departing from the
spirit and scope of the present invention.
* * * * *